154 kriegsfeld et al

7/31/2019 154 kriegsfeld et al

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Review

The regulation of neuroendocrine function: Timing is everything

Lance J. Kriegsfeld a,, Rae Silverb,c,d

a Department of Psychology and Helen Wills Neuroscience Institute, 3210 Tolman H all, #1650, University of California, Berkeley, CA 94720-1650, USAb Department of Psychology, Barnard College, New York, N Y 10027, USA

c Department of Psychology, Columbia University, New York, NY 10027, USAd Department of Anatomy and Cell Biology, College of Physicians and Surgeons, New York, NY 10032, USA

Received 24 September 2005; revised 6 December 2005; accepted 8 December 2005

Available online 21 February 2006

Abstract

Hormone secretion is highly organized temporally, achieving optimal biological functioning and health. The master clock located in the

suprachiasmatic nucleus (SCN) of the hypothalamus coordinates the timing of circadian rhythms, including daily control of hormone secretion. In

the brain, the SCN drives hormone secretion. In some instances, SCN neurons make direct synaptic connections with neurosecretory neurons. In

other instances, SCN signals set the phase of clock genes that regulate circadian function at the cellular level within neurosecretory cells. The

protein products of these clock genes can also exert direct transcriptional control over neuroendocrine releasing factors. Clock genes and proteins

are also expressed in peripheral endocrine organs providing additional modes of temporal control. Finally, the SCN signals endocrine glands via

the autonomic nervous system, allowing for rapid regulation via multisynaptic pathways. Thus, the circadian system achieves temporal regulation

of endocrine function by a combination of genetic, cellular, and neural regulatory mechanisms to ensure that each response occurs in its correct

temporal niche. The availability of tools to assess the phase of molecular/cellular clocks and of powerful tract tracing methods to assess

connections between clock cells and their targets provides an opportunity to examine circadian-controlled aspects of neurosecretion, in the

search for general principles by which the endocrine system is organized.

2005 Elsevier Inc. All rights reserved.

Keywords: Circadian; Diurnal; Endocrinel; Neurosecretion; Clock genes; Suprachiasmatic

Contents

Circadian aspects of reproduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558

Circadian control of endocrine secretions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559

Endocrine influences on the circadian system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559

Identification of a brain clock: from tissue to gene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560

Circadian output and orchestration of endocrine function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560

Diffusible signals controlling behavioral rhythms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560

Neural control of neurosecretory factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563

Neural SCN output and estrus regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563Direct and indirect transcriptional control as a clock output . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565

Direct transcriptional control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565

Indirect transcriptional control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566

System-level control and coordination of endocrine function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566

Clocks in the neuroendocrine system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567

The top of the hierarchy: neural SCN output . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567

Hormonal and neural communication to glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567

Hormones and Behavior 49 (2006) 557574www.elsevier.com/locate/yhbeh

Corresponding author. Fax: +1 510 642 5293.

E-mail address: [email protected] (L.J. Kriegsfeld).

0018-506X/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.yhbeh.2005.12.011
mailto:[email protected]:[email protected]://dx.doi.org/10.1016/j.yhbeh.2005.12.011http://dx.doi.org/10.1016/j.yhbeh.2005.12.011http://dx.doi.org/10.1016/j.yhbeh.2005.12.011mailto:[email protected]


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Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569

Circadian aspects of reproduction

The importance of circadian (about a day) timing in

hormone production/secretion has been known since the

1950s when Everett and Sawyer determined that a

stimulatory signal occurring during a narrow temporal

window on the afternoon of proestrus is necessary for

induction of ovulation later that night (Everett and Sawyer,

1950). Such close temporal organization is important for

successful reproduction, as numerous hormone-dependent

behavioral and physiological processes must be coordinated.

If optimal temporal relationships are disrupted, pronounced

deficits in fertility can result. For example, ovulation,

behavioral estrus, fertilization, and pregnancy maintenancerequire a specific temporal pattern of hormone secretion in

spontaneous ovulators such as rats, hamsters, and mice

(Blaustein et al., 1994; McEwen et al., 1987; Mong et al.,

2003). Prior to behavioral estrus, rising levels of estrogen

both trigger a precisely timed preovulatory surge in

luteinizing hormone (LH) and stimulate the production of

brain progesterone receptors in preparation for progesterone

effects on neurons. The timing of progesterone receptor

regulation relative to estrogen ensures that behavioral

receptivity is coordinated with the time of ovulation,

thereby increasing the likelihood of pregnancy (Hansen et

al., 1979). Following ovulation, a prolactin surge is

necessary to support the corpus luteum to maintainprogesterone secretion necessary for pregnancy and its

maintenance (Egli et al., 2004). This sequence is under

circadian control.

In the present review, we summarize evidence indicating that

the timing of endocrine secretions is coordinated by a brainclock located in the suprachiasmatic nucleus (SCN) of the

hypothalamus (Fig. 1). Over the last decade, there have been

substantial, rapid advances in our understanding of the circadian

modulation of brain and peripheral organ activity. Current data

indicate that a neural signal from the SCN is necessary for the

circadian timing of hormone secretion, while a diffusible signal

is sufficient to modulate non-endocrine events such as daily

behavioral activities. The identification of core clock genes,

clock-controlled genes (CCG), and their localization in

neurosecretory cells (Kriegsfeld et al., 2003; Olcese et al.,

2003), the pituitary gland (Shieh, 2003; Von Gall et al., 2002)

and a number of peripheral endocrine glands (Bittman et al.,2003; Morse et al., 2003; Zylka et al., 1998) each provide new

opportunities for evaluating the loci and mechanisms of

temporal gating of hormone secretion.

We review evidence that hormone secretion is regulated not

only by the feedback loops long studied by endocrinologists but

also by the SCN and SCN-derived temporal signals acting

directly on neurosecretory cells, on the autonomic nervous

system, and on clock genes and clock-controlled genes. To this

end, we present an abbreviated introduction to the core negative

feedback loop controlling cellular circadian clock function to

provide a basis for understanding how time can be tracked

within a cell and to set the foundation for understanding the

possible role of clock genes in endocrine regulation. Fordetailed summaries of research on cellular/molecular clock

genes and proteins, the reader is referred to the following

reviews (Albrecht, 2004; Ashmore and Sehgal, 2003; Du and

Fig. 1. The mammalian circadian clock is located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. The SCN pictured here in this schematic is a

coronal section through a rodent brain. The SCN is situated at the base of the brain of the brain directly above the optic chiasm (oc) and surrounding the third ventricle(V3). The sagittal schematic in the upper right corner depicts the approximate rostralcaudal location depicted in the coronal section.

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Tong, 2002; Duffield, 2003; Gachon et al., 2004; Glossop and

Hardin, 2002; Green, 2003; Hastings et al., 2003; Liu, 2003;

Okamura, 2003a,b; Okamura et al., 2002; Piggins, 2002;

Roenneberg and Merrow, 2003; Schibler and Sassone-Corsi,

2002; Schultz and Kay, 2003; Zordan et al., 2003).

Circadian control of endocrine secretions

Biological events typically exhibit marked, predictable

cycles ranging in time from seconds to years. In the endocrine

system, virtually every factor measured to date shows a

circadian (endogenous) or diurnal (driven) rhythm. These

alterations are achieved by modulation of pulse amplitude

(i.e., amount of hormone released), pulse frequency (i.e., rate of

hormone release), or by a combination of both of these

processes. For example, studies in male rhesus macaques in

which animals were sampled at 20-min intervals in an LD cycle

revealed diurnal rhythms in luteinizing hormone (LH),

testosterone, prolactin, and cortisol (Plant, 1981). Likewise,studies in rats, Syrian hamsters, and humans indicate circadian

variation in gonadotropins and gonadal steroids around the

onset of puberty (Andrews and Ojeda, 1981; Boyar et al., 1976;

de la Iglesia et al., 1999; Jakacki et al., 1982; Smith and Stetson,

1980). It has been suggested that diurnal variation in hormone

concentrations may simply be modulated by sleep. However,

sleep reversal (subjects sleep during the day rather than night)

and sleep interruption do not affect the daily pattern of most

hormones, confirming regulation by an endogenous clock

independent of sleep (Desir et al., 1982; Kapen et al., 1974; Van

Cauter and Refetoff, 1985), although interactions between sleep

and the circadian system exist (Kriegsfeld et al., 2002a). The

present review highlights the current understanding of circadiansystem regulation of endocrine rhythms via multiple means of

modulation and proposes a novel mechanism of hierarchical

control beginning with the master brain clock. For a complete

description of daily hormone secretion patterns, the reader is

referred to the following reviews (Gore, 1998; Hastings, 1991;

Kriegsfeld et al., 2002a; Turek and Van Cauter, 1994).

Endocrine influences on the circadian system

Not only does the SCN regulate endocrine rhythms but

hormones also feed back to the SCN, presumably to fine-

tune the temporal pattern of endocrine secretion givencurrent conditions. For example, high-affinity melatonin

receptors are localized to the SCN, and administration of

melatonin can alter SCN phase (Dubocovich et al., 1996;

Hastings et al., 1997; Lewy et al., 1992; Slotten et al., 1999;

Vanecek and Watanabe, 1999). In addition, exogenous

melatonin alters the phase of SCN electrical activity measured

in a slice preparation in vitro in a manner predicted by the

phase response curve (McArthur et al., 1991). The sensitivity

of the SCN to melatonin may be a function of daily variation

in the density of melatonin binding sites within the SCN

(Schuster et al., 2001). In humans, melatonin administration

causes phase delays during late night or early morning and

phase advances in late morning to early afternoon (Lewy and

Sack, 1997). This finding has significant implications for shift

workers, jet lag, and the blind.

Estrogen has pronounced effects on circadian activity

rhythms, likely through both direct effects on the SCN and

indirect mechanisms. In females, estrogen modulates both

period and activity consolidation, suggesting actions on the

circadian clock rather than transient effects on SCN targets.Cycling female hamsters and rats show a phase advance in

locomotor activity on the day of estrous (scalloping), when

estradiol levels are highest, and continuous administration of

estradiol in silastic capsules shortens the free-running period of

ovariectomized hamsters (Morin et al., 1977). When hamsters

are maintained in constant light, the normally stable activity

phase frequently splits into two activity components that

stabilize approximately 12 h apart. Continuous administration

of estradiol in silastic capsules to ovariectomized hamsters

prevents these changes (Morin, 1980).

Direct effects of estrogen on the circadian clock are

suggested by the expression of and estrogen receptors inthe SCN across mammals, including humans (Gundlah et al.,

2000; Kruijver and Swaab, 2002; Su et al., 2001). These

receptors may be important for its normal development and

synchronization to the environment (Abizaid et al., 2004;

Gundlah et al., 2000). In humans, the presence of estrogen

receptor expression, along with sex differences in SCN

structure, suggests that estrogen may act on the SCN during

development (Hofman et al., 1988, 1996; Kruijver and Swaab,

2002). Indirect effects in estrogen on the circadian clock are

indicated by studies in which simultaneous injection of

anterograde and retrograde tract tracers into the SCN reveal

that ER-expressing cells in the preoptic area, amygdala,

BNST, and arcuate provide input to the SCN, but the SCN doesnot project directly to these ER-expressing cells (de la Iglesia

et al., 1999).

In males, testosterone also affects consolidation of locomotor

activity rhythms. Extended exposure to short day lengths

induces a decrease in testicular size and a decline in plasma

testosterone concentrations in male hamsters (Ellis and Turek,

1983). Following testicular regression (or after castration), there

is an increase in lability of activity onset, an expansion of the

daily activity duration, with a decrease in wheel revolutions per

cycle; testosterone replacement prevents these changes (Morin

and Cummings, 1981). Testosterone may act through SCN

androgen receptors. To date, androgen receptors have beenidentified in the SCN of several species (Clancy et al., 1994;

Fernandez-Guasti et al., 2000; Kashon et al., 1996; Michael and

Rees, 1982; Rees and Michael, 1982). Alternatively, testoster-

one may exert its effects through conversion to estradiol, which

may act either directly on receptors in the SCN (e.g., Shughrue

et al., 1997), or indirectly in ER-expressing cells in other brain

areas that, in turn, communicate with the SCN (de la Iglesia et

al., 1999). In rats, conversion of testosterone to estradiol may be

important for the activity-stimulating effects of testosterone

(Roy and Wade, 1975). Estradiol is nearly 100 times as effective

as testosterone at increasing activity, while dihydrotesosterone

(a non-aromatizable androgen) has no effect on wheel-running

activity of rats (Roy and Wade, 1975). Taken together, these

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findings suggest that the conversion of testosterone to estrogen,

by aromatase, may be important for the effects of testosterone

on circadian rhythms.

Identification of a brain clock: from tissue to gene

A highly localized brain clock in mammals was firstsuggested in 1972, with the demonstration that lesions ablating

the SCN abolish circadian rhythmicity in adrenal corticoid

secretion and locomotor behavior (Moore and Eichler, 1972;

Stephan and Zucker, 1972). SCN-lesioned animals continue to

show the full range of normal behaviors, but their temporal

organization is lost and never recovers, irrespective of how early

in development the lesions are performed (Mosko and Moore,

1979). The initial conclusion that the SCN serves as a brain

master clock has been confirmed in the subsequent 30 years by

converging lines of research involving in vivo, ex vivo, and in

vitro studies carried out in many different laboratories. For

example, transplants of donor SCN tissue into the brains ofarrhythmic, SCN-lesioned hosts restore circadian rhythmicity in

behavior (Lehman et al., 1987; Ralph et al., 1990). Importantly,

rhythms are restored with the period of the donor SCN,

indicating that the transplanted tissue does not act by restoring

host brain function but that the clock is contained in the

transplanted tissue. Further evidence that clock function is

contained within the SCN comes from studies demonstrating

that circadian rhythms in neural firing rate persist in isolated

SCN tissue maintained in culture (Green and Gillette, 1982;

Groos and Hendriks, 1982; Shibata et al., 1982). An excellent

overview of these studies in historical perspective is available

(Weaver, 1998).

While the foregoing work demonstrated that the SCN tissueas a whole served as a clock, the finding that circadian

rhythmicity is a property of individual SCN neurons set the

stage for the next breakthrough. The demonstration that

dispersed, cultured SCN cells exhibit circadian rhythms of

electrical activity indicated that circadian timing is a cellular

property rather than an emergent property of a neural network

(Welsh et al., 1995). These studies allowed for the exploration

and subsequent discovery of the cellular molecular machinery

responsible for circadian function.

Within a cell, circadian rhythms are produced by an

autoregulatory transcriptional/translational negative feedback

loop that takes approximately 24 h, whereas the generalmechanism for circadian oscillations at the cellular level is

common among organisms, the components comprising the

feedback loop differ. For the purpose of clarity, only the core

mammalian feedback loop is described. To date, it is thought

that two proteins, CLOCK and BMAL1, bind to one another

and drive the transcription of messenger RNA (mRNA) of the

Period (Per) and Cryptochrome (Cry) genes by binding to the

E-box (CACGTG) domain on these gene promoters. Three

Period (Per1, Per2, and Per3) and two cryptochrome genes

(Cry1 and Cry2) have been identified. The mRNA for these

genes is translated into PER and CRY proteins in the cytoplasm

of the cell over the course of the day. Throughout the day, these

proteins build up within the cytoplasm, and when they reach

high enough levels, they form hetero- and homo-dimers. These

newly formed dimers then feed back to the nucleus where they

bind to the CLOCK:BMAL1 protein complex to turn off their

own transcription (Fig. 2). Numerous otherclock genes and

regulatory enzymes have been identified but will not be

reviewed for the sake of brevity. Future studies on the specific

genes and their interactions that result in circadian timekeepingat the cellular level will likely yield exciting new information on

other regulatory elements and their interactions.

Discovery of the genes regulating circadian rhythmicity led

to breakthroughs identifying clock genes and their protein

products in numerous sites, including extra-SCN brain loci and

in the periphery (Abe et al., 2002; Balsalobre et al., 1998;

Kriegsfeld et al., 2003; Yamazaki et al., 2000). These findings,

in turn, led to questions about the unique nature of the master

oscillator in the SCN, the functional significance of extra-SCN

oscillators, and mechanisms of coordination of these widely

dispersed clocks.

To compare cellular mechanisms of clock gene expression inthe SCN and in the periphery, embryonic fibroblasts from wild-

type and (behaviorally arrhythmic) Cry/ mice were used

(Yagita et al., 2001). Clock properties of cell lines derived from

peripheral cells of each strain were similar to those of the strain-

specific SCN, supporting the conclusion of common core clock

gene function in all tissues (Yagita et al., 2001). It remains

controversial, however, whether peripheral oscillators are

similar to those of the SCN in their ability to sustain endogenous

rhythmicity for long durations (Balsalobre et al., 1998; Yoo et

al., 2004).

When a tissue, either SCN or peripheral, loses coherent

rhythmicity, it is important to determine whether this is due to

dampening of rhythms in individual cells or to loss ofsynchrony among a population of cells in the tissue. Use of

Per1-luciferase transgenic animals indicates that rhythms in

peripheral tissues damp then disappear over time due to

uncoupling (desynchronization) among oscillators that retain

their individual rhythms (Nagoshi et al., 2004; Welsh et al.,

2004). Presumably peripheral clock cells normally get phase

information (directly or indirectly) from the SCN to synchronize

individual oscillators to each other. In this view, the SCN sets

the phase of peripheral circadian clocks daily, coordinating the

activity of tissues and organs of the body relative to one another,

thereby maintaining homeostasis.

Circadian output and orchestration of endocrine function

Diffusible signals controlling behavioral rhythms

Rhythmic electrical activity and oscillation of clock genes

within the SCN neurons ultimately lead to rhythmicity in the

whole organism. Compelling evidence for a diffusible output

signal derives from neural tissue transplantations in which the

SCN from a fetal donor is implanted into the third ventricle of

an adult, SCN-lesioned host. As mentioned previously, these

grafts restore activity-related behaviors such as locomotor,

drinking, and gnawing rhythms (Lehman et al., 1987; Ralph et

al., 1990; Silver et al., 1990). That a diffusible signal is



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sufficient to restore locomotor rhythmicity in SCN-lesioned

hosts was demonstrated by encapsulating donor SCN tissue in a

membrane that prevented neural outgrowth while allowing the

diffusion of signals between graft and host (Silver et al., 1996).

One candidate diffusible signal is prokineticin-2 (PK2)(Cheng et al., 2002). This protein is expressed rhythmically in

the SCN, and its receptor is present in all major SCN targets

(Cheng et al., 2002, 2005). Likewise, PK2 administration

during the night (when levels are low) inhibits wheel running

behavior. Whether or not this signal normally operates in a

diffusible manner and/or is released synaptically requires

further examination. A second candidate diffusible signal is

transforming growth factor-alpha (TGF-alpha) (Kramer et al.,

2001). As with PK2, TGF-alpha is expressed rhythmically in

the SCN, and its administration inhibits wheel-running

behavior. The receptor for TGF-alpha is also expressed in the

SPVZ, the major target of the SCN. The degree which TGF-

alpha is released in a diffusible manner under normal conditions

is not known. Studies in which the contribution of neural

efferents and diffusible signals can be distinguished are

necessary to begin to answer this question.

Although it is intriguing to speculate on the role of these

signals in communicating information from the SCN, theproblem of unequivocally identifying an endogenous, physio-

logically relevant diffusible SCN signal is complex and parallel

in scope to the task faced by Sir Geoffrey Harris' in providing

evidence for hypothalamic control over pituitary function in the

1950s. The necessary and sufficient criteria to confirm the

existence of a diffusible signal in a fluid volume have been

summarized previously (Nicholson, 1999). First, the removal or

replacement of the signaling substance must result in a change

in the response being controlled. The substance should be

present or increased, or both, in a well-defined temporal

relationship to the response (and similarly declines when the

response disappears). In addition, evidence must be obtained

that a fluid compartment is the conduit for a diffusible or

Fig. 2. A simplified model of the intracellular mechanisms responsible for mammalian circadian rhythm generation. The process begins when CLOCK and BMAL1

proteins dimerize to drive the transcription of the Per(Per1, Per2, and Per3) and Cry (Cry1 and Cry2) genes. In turn,Perand Cry are translocated to the cytoplasm and

translated into their respective proteins. Throughout the day, PER and CRY proteins rise within the cell cytoplasm. When levels of PER and CRY reach a threshold,

they form heterodimers, feed back to the cell nucleus and negatively regulate CLOCK:BMAL1 mediated transcription of their own genes. This feedback loop takes

approximately 24 h, thereby leading to an intracellular circadian rhythm.



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transported signal. The signal must have access to and enter the

compartment where the fluid dynamics and turnover in the

compartment should allow appropriate movement of the signal.

While PK2 and TGF-alpha meet some of these criteria, further

research is necessary to clarify the role of these molecules in

communicating circadian information.

Neural control of neurosecretory factors

In contrast to behavioral rhythms (e.g., locomotion, drinking,

gnawing), endocrine rhythms require neural projections from

the SCN to endocrine targets; endocrine rhythms are abolished

after knife cuts severing SCN efferents (Hakim et al., 1991;

Nunez and Stephan, 1977) and are not restored in SCN-lesioned

transplanted animals (Meyer-Bernstein et al., 1999; Nunez and

Stephan, 1977; Silver et al., 1996), presumably due to

inadequate neural innervation of the host brain by the graft.

Further evidence for a neural SCN output signal regulating

hormone secretion is seen in studies of female hamsters. Whenhoused in constant light, the activity of a subset of hamsters

splits into two separate activity bouts within a 24-h interval.

These split females display two daily LH surges, each

approximately half the concentration of a single surge in a

non-split female (Swann and Turek, 1985) (Fig. 3). Under

normal conditions, both halves of the bilaterally symmetrical

SCN are active in synchrony. In ovariectomized, estrogen-

implanted split hamsters killed during one of their activity

bouts, however, activation of the SCN occurs on one side of the

brain (monitored by FOS expression) but not on the other,

suggesting that each half of the SCN can control an activity bout

(de la Iglesia et al., 2000). Remarkably, FOS activation in

GnRH neurons was only seen on the side of the brain in whichSCN FOS expression occurred (de la Iglesia et al., 2003). These

findings suggest that the precise timing of the LH surge is

derived from a neural signal originating in the SCN and

communicated to ipsilateral GnRH neurons, as a diffusible

output signal would reach both sides of the brain. Importantly,

some hypothalamic sites are activated ipsilaterally, while others

are activated either ipsilaterally or bilaterally in the split animal,

again supporting the notion of multiple SCN output pathways

(Tavakoli-Nezhad and Schwartz, 2005; Yan et al., 2005).

Neural output from the SCN has been extensively

investigated in rats and hamsters using tract tracing

techniques (Kalsbeek et al., 1993; Kriegsfeld et al., 2004;Leak and Moore, 2001; Morin et al., 1994; Stephan et al.,

1981; Watts and Swanson, 1987; Watts et al., 1987).

Importantly, many of these monosynaptic projections target

brain regions containing neuroendocrine cells producing

hypothalamic releasing hormones (Fig. 4). Direct projections

have been traced from the SCN to the medial preoptic area

(MPOA), supraoptic nucleus (SON), anteroventral periven-

tricular nucleus (AVPV), the paraventricular nucleus (PVN),

the dorsomedial nucleus of the hypothalamus (DMH), and the

lateral septum and the arcuate (Arc). The SCN also projects to

the pineal through a multisynaptic pathway (Klein, 1985;

Klein et al., 1983). There is abundant evidence for direct

neural SCN control of neuroendocrine cell populations (Buijs

et al., 1998, 2003; Egli et al., 2004; Gerhold et al., 2001;Horvath, 1997; Horvath et al., 1998; Kalsbeek and Buijs,

2002; Kalsbeek et al., 1996a,b, 2000; Kriegsfeld et al., 2002a,

b; Van der Beek et al., 1993, 1997b; Vrang et al., 1995).

Because these cell populations can regulate neurochemicals

that are secreted into the CSF (Reiter and Tan, 2002; Skinner

and Caraty, 2002; Skinner and Malpaux, 1999; Tricoire et al.,

2003) or general circulation, SCN-derived signals can control

widespread systems in the brain and body.

Together, the findings summarized above suggest several

possibilities: behavioral rhythms may be controlled by a

diffusible signal(s), while endocrine rhythms may require

neural output. Alternatively, behavioral and endocrine rhythmscan both be supported by diffusible signals, but the threshold for

supporting behavioral rhythms is lower. Finally, behavioral

rhythms are controlled by both neural and diffusible signals, and

either can maintain rhythmic function, while endocrine rhythms

can only be supported via neural connections. Definitive

identification of biologically significant endogenous diffusible

signal(s) and the precise mode of SCN control is a current line

of inquiry.

Neural SCN output and estrus regulation

SCN control of the rodent estrous cycle has been investigated

extensively (Barbacka-Surowiak et al., 2003), providing anexcellent model system for investigations of circadian and

neuroendocrine interactions. The SCN sends projections

directly to GnRH neurons in female rodents (Horvath et al.,

1998; Van der Beek et al., 1997a). These efferents express the

SCN peptide, vasoactive intestinal polypeptide (VIP). GnRH

neurons particularly important for the regulation of the estrous

cycle are activated at the time of proestrus and receive SCN

input (van der Beek et al., 1994). Also, sex differences in the

daily expression of SCN VIP mRNA are seen in rats, with

females exhibiting a peak 12 h out of phase with that of males

(Krajnak et al., 1998). Presumably, the signal regulating the

estrous cycle is sexually dimorphic, thereby lending furthersupport for VIP regulation of estrus. Furthermore, antisense

oligonucleotides directed against VIP lead to a delayed and

attenuated LH surge (Harney et al., 1996), reminiscent of that

seen in middle-aged rats (Gore, 1998). Sex differences also exist

in the pattern of projections from the SCN (Horvath et al., 1998;

Van der Beek et al., 1997a,b). These sex differences in SCN

projections upon GnRH cells, and in the production of

Fig. 3. Circadian control of gonadotropin secretion. Syrian hamsters normally exhibit one consolidated bout of activity every 24 h. Under conditions of constant light,

the hamsters activity splits intotwo components separated by about 12 h. In one ingenious study(de la Iglesia et al., 2003), the investigators killed animals prior to each

of the activity bouts (see asterisk on activity records). Brains were analyzed for FOS activity in the SCN and in neurons of the GnRH neuronal system. Splitting

behavior resulted in only one half of the SCN being active during a given time of day. GnRH was only activated (expressed FOS) on the side of the brain in which the

SCN was active. Given that ovariectomized, estrogen-implanted hamsters with split behavior experience two LH surges (Swann and Turek, 1985), we are speculatingthat the neural mechanism underlying this phenomenon can be explain by differential leftright activation of the GnRH system at two times of day.



8/18

neurochemicals in SCN cells projecting to the GnRH system,

may underlie the absence of an LH surge in males.In addition to direct connections to GnRH neurons, the SCN

projects extensively to the anteroventral periventricular nucleus

(AVPV), a brain region associated with the induction of the

preovulatory LH surge (Le et al., 1997; Levine, 1997). The cells

to which the SCN projects are estrogen-responsive (Watson et

al., 1995), suggesting that the AVPV may be an important

integration point for circadian and steroidal signals. Given the

widespread projections of the SCN throughout the CNS, along

with extensive input to the GnRH system, the potential for

additional indirect modulation of the reproductive axis is

considerable.

Vasopressin, another SCN peptide important in the regula-

tion of the estrous cycle, is synthesized and released in acircadian manner. SCN vasopressin is rhythmically secreted

with a peak during a sensitive time window prior to the LH

surge (Kalsbeek et al., 1996a,b). Vasopressin administration

into the MPOA induces an LH surge in SCN-lesioned,

ovariectomized rats treated with estradiol (Palm et al., 2001a,

b). Electrical stimulation of the MPOA and VP administration

into the MPOA induces an LH surge in SCN-lesioned rats

(Palm et al., 1999). Finally, in co-cultures of POA and SCN

tissue, the rhythm of GnRH release is in phase with the rhythm

of VP release, but not with that of VIP (Funabashi et al., 2000).

Paradoxically, Brattleboro rats incapable of synthesizing

vasopressin are fertile, although abnormal estrous cyclicityand reduced fertility have been noted (Boer et al., 1982). Taken

together, these data indicate a potential role for VP in inducing

the LH surge, although it is likely not the sole mediator.

Positive feedback effects of estrogen serve a permissive role

in initiating the LH surge upon the arrival of the signal from the

circadian pacemaker (Barbacka-Surowiak et al., 2003; Levine,

1997); implants of an anti-estrogen into the POA block the LH

surge (Petersen and Barraclough, 1989). This dependence on

estrogen ensures the maturation of the follicle during the time of

the surge, while the circadian dependence ensures that receptive

behavior coincides with ovulation. It remains unclear, however,

how these two signals converge at the cellular level to allow

integration at the appropriate time of day. One possibility is that

estrogen alters neurochemical secretion by the SCN or the

signaling efficacy of these chemicals via second messengersystems and kinases (Chappell and Levine, 2000; Levine, 1997).

An alternative hypothesis is that estrogen stimulates ligand-

independent progesterone receptor production, and the timed

neuronal signal acts on progesterone receptors (Chappell et al.,

2000; Levine, 1997; Levine et al., 2001). This scheme suggests

that the effects of estrogen are integrated with the SCN signal at

the level of progesterone receptors to ensure that the GnRH

system is sensitive to the daily signal only during the

preovulatory estrogen surge. The fact that few estrogen receptors

have been localized to GnRH neurons (Shivers et al., 1983)

suggests that estrogen acts to produce progesterone in neurons

upstream of the GnRH system and hints at important,

unidentified, SCN projections to these upstream components.Given the potential importance of estrogen/progesterone

receptor expressing neurons upstream of the GnRH system, it

will be interesting to establish the means by which the circadian

system communicates with and modulates these regulatory

elements.

The importance of a functional molecular clock in driving

GnRH cells is seen in mice with a mutant form of the Clock

gene. These mice have long, irregular estrous cycles and fail to

exhibit an LH surge following estradiol treatment. Furthermore,

this mutation also leads to an increase in fetal reabsorption

during pregnancy and a decline in full-term parturition (Miller

et al., 2004). These deficits are associated with a decline in mid-term levels of progesterone, suggesting abnormal secretion

patterns of prolactin (Miller et al., 2004). Together, these

findings highlight the importance of the circadian system in

regulating the temporal pattern of hormone secretion necessary

for mating, pregnancy, and its maintenance, although results

using mutant models must be interpreted cautiously until

converging approaches consistently support these conclusions.

Not only does the SCN regulate GnRH during the estrous

cycle, but other aspects of the estrous cycle are also regulated by

the SCN. During proestrus, rising levels of estradiol reach a

critical point and trigger the release of prolactin at a specific

time of day. This release of prolactin is dependent upon the

estradiol-induced increase in tuberoinfundibular dopaminergic

Fig. 4. Efferent projections of the rodent SCN to its targets in the brain. Below each target area is a list of neuroendocrine cells that lie in that region of the brain and

could potentially be regulated by direct projections from the SCN. Solid lines represent monosynaptic projections, while the dotted line represents a multisynaptic

projection to the pineal gland. The pronounced overlap between neuroendocrine cells and SCN efferent terminals, combined with reports demonstrating direct

neuronal projections from the SCN to neuroendocrine cells (e.g., GnRH and CRH cells), provides suggestive evidence for a global mechanism of circadian hormonal

regulation. Adapted from Kriegsfeld et al. (2002a) with permission.



9/18

(TIDA) neuron activity (Neill et al., 1971). Administration of an

estradiol antiserum on the morning of diestrus-2 blocks the

proestrous surge of prolactin (Neill et al., 1971). The proper

timing of prolactin is achieved via SCN projections to TIDA

neurons in the arcuate (Horvath, 1997). Additionally, TIDA

neurons rhythmically express the clock gene, Per1, providing a

potential additional means of temporal control (Kriegsfeld et al.,2003)and see below). SCN lesions result in an abolition of a

daily prolactin rhythm (Mai et al., 1994) and abolish the

preovulatory prolactin surge (Pan and Gala, 1985). Given the

importance of prolactin timing in maintaining the corpus

luteum, these findings further suggest an essential role for the

circadian clock in reproduction and may explain the increased

fetal reabsorption in Clockmutant mice described above (Miller

et al., 2004).

It has been well established that environmental factors act to

fine-tune endogenous regulation of the reproductive cycle. For

example, the timing of the prolactin surge is regulated by the

phase of the light

dark (LD) cycle. A change in the LD cycleleads to predictable changes in the timing of the estrogen-

induced prolactin surge and the mating-induced prolactin surge

(Blake, 1976; Pieper and Gala, 1979). Thus, environmental time

of day information is transmitted to the circadian system to

precisely coordinate reproductive events relative to local time.

Cervically stimulated prolactin surges have a free-running cycle

in ovariectomized, estradiol-treated rats held in constant

conditions. This pattern is abolished after ablation of the SCN

(Bethea and Neill, 1980). As with the LH surge, this finding

suggests that both the timing and production of the prolactin

surge require a signal from the circadian clock. The means by

which environmental stimuli other than light (e.g., social signals,

local conditions, nutrition, etc.) are integrated into this system tofine-tune the timing of hormonal and behavioral events are

unknown and represent an opportunity for exploration.

As with estrous behavior, the preovulatory LH surge

occurs at regular 4- or 5-day intervals in rats, on the day of

proestrus at a specific time of day coupled to the LD cycle

(Colombo et al., 1974). Interestingly, despite the fact that the

temporal pattern of SCN neural activity is similar in nocturnal

and diurnal rodents (Smale et al., 2003), with a peak during

the day and a trough at night, the timing of the preovulatory

LH surge is reversed (Mahoney et al., 2004; McElhinny et al.,

1999). Although the mechanisms by which this reversal

occurs remain elusive, comparisons between diurnal andnocturnal species may provide insight into how circadian

control is accomplished in humans (i.e., a diurnal species).

Studies in rhesus initially indicated that the LH surge could

be induced at any circadian phase in primates (Knobil, 1974).

However, frequent urinary LH monitoring of women with

regular menstrual cycles suggests a pronounced influence of

circadian timing on the preovulatory LH surge, with most

exhibiting the LH surge between midnight and 8:00 AM

(Cahill et al., 1998; Edwards, 1981) (Fig. 5). Of 155 regular

cycles monitored, 146 surges occurred during this 8-h time

window (Cahill et al., 1998). Given that the daily timing of

the LH surge in women can be unmasked under carefully

monitored and controlled conditions, it is likely that the

human ovulatory cycle also requires interactions between the

circadian and reproductive systems.

Direct and indirect transcriptional control as a clock output

The circadian system exerts a widespread influence over

numerous bodily functions. DNA microarray studies in mice

indicate that59% of the genome, excluding genes involved

in the core clock loop, are under circadian control (Akhtar et al.,

2002; Panda et al., 2002; Storch et al., 2002). However, these

so-called clock-controlled genes (CCGs) differ among tissues,

with any two tissues likely sharing less than 10% of CCGsunder circadian control. Together, these findings suggest that

circadian control is ubiquitous throughout the body, and tissue-

specific processes may be controlled by differential activation

of downstream genes in individual systems.

Direct transcriptional control

CCGs maintain a predictable phase relationship with the core

clock genes (Ueda et al., 2002), indicating that the CCGs are

either directly or indirectly regulated via the circadian

transcriptional machinery. The expression of some CCGs is

directly controlled by the CLOCK:BMAL1 heterodimer bindingto an E-box enhancer (CACGTG) in their promoter (Jin et al.,

1999). An example of direct transcriptional control by the

circadian system is seen with vasopressin regulation. Vasopres-

sin is present in the SCN where it acts locally to regulate rhythm

generation (Mihai et al., 1994a,b). It cannot be the sole

regulatory factor, as the SCN of Brattleboro rats maintains

rhythms in electrical activity (Ingram et al., 1998), and these

animals exhibit only slight disruptions in rhythm amplitude and

entrainability (Brown and Nunez, 1989; Murphy et al., 1993,

1996). In addition to a role within the nucleus, SCN,

vasopressin-expressing neurons signal distant hypothalamic

targets and SCN vasopressin signaling has been implicated in

the control of estrus (Buijs et al., 2003a,b; Kalsbeek et al., 1996a,

Fig. 5. Frequency of onset of the LH surge by time of day. A total of 155 cycles

from 35 women were monitored. The graph represents the percentage of

preovulatory LH surges occurring during each time interval. Adapted from

Cahill et al. (1998).



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b; Mihai et al., 1994a,b; Palm et al., 2001a,b). The SCN rhythm

in vasopressin (a rhythm that is not present in other

vasopressinergic cell populationsee below) is dependent on

an E-box element in the 5 flanking region of the vasopressin

gene to which the CLOCK:BMAL complex binds. The

vasopressin rhythm is abolished in mutant mice with aberrant

Clock gene expression (Jin et al., 1999; Silver et al., 1999).E-box elements in the promoter have the potential for direct

control by circadian clock genes, but this alone is not sufficient.

In the SON (unlike the SCN), vasopressin is dependent on

osmotic balance and is not rhythmic (Jin et al., 1999). While

expression of Clock is robust in the SCN and SON, Bmal1 is

expressed robustly only in the SCN and is barely detectable in

the SON. These findings indicate some of the conditions

necessary for direct control of genes regulating neuroendocrine

function by the circadian clock transcriptional machinery.

A gene with an E-box in its promoter region is a potential

target for direct control by the transcriptional machinery of the

circadian clock. We screened hypothalamic and pituitaryendocrine factors for E-box elements in the promoter of their

published gene sequences to determine their potential for this

means of control (Table 1). We found that some releasing

hormones (TSH, GHRH, and vasopressin) do have E-box

enhancers. We have shown in the adult mouse brain that a

subset of neuroendocrine cells in the preoptic area, paraven-

tricular nucleus of the hypothalamus, and the arcuate nucleus

express the clock gene Per1 (Kriegsfeld et al., 2003), suggesting

that this direct transcriptional regulation likely plays a key role

in the organization of endocrine timing.

Indirect transcriptional control

In studies of the liver, it has been demonstrated that D-

element binding protein (DBP) is another CCG under direct

transcriptional regulation by the core circadian feedback loop

(Ripperger et al., 2000). Importantly, DBP binds to other gene

promoters to temporally regulate their transcription. This

process is important in the control of hepatic metabolic

processes; DBP activates the transcription of albumin, choles-

terol 7 hydroxylase, and cytochrome P450 (Lavery et al.,

1999). This second order system can provide ubiquitous control

via the circadian system and temporal control of key enzymatic

pathways involved in hormone production.

A process similar to hepatic regulation by DBP may

modulate the reproductive axis, as the gene for GnRH doesnot have an E-box but appears to be under circadian control. A

series of studies using GT1 cells, an immortalized line of GnRH

cells, demonstrated the rhythmic expression of numerous

circadian clock genes (Chappell et al., 2003; Gillespie et al.,

2003; Olcese et al., 2003). Importantly, GnRH release occurs

episodically approximately every 90 min in most species, and

GT1 cells also express this ultradian pattern of GnRH

production. When clock genes are disrupted in GT1 cells, not

only is the daily rhythm disrupted, but both pulse frequency and

amplitude are also dramatically altered (Chappell et al., 2003).

These findings suggest that, in addition to 24-h cycles, clock

genes expressed within neuroendocrine cells may regulate theepisodic pattern of hormone secretion outside of the circadian

range, even when the regulated neuroendocrine gene lacks an E-

box enhancer. While these data are intriguing, a direct link

between clock genes and ultradian rhythmicity awaits further

supporting evidence.

While the forgoing studies using embryonic cell lines

provide insights into cellular mechanisms, how these data

generalize to functioning in vivo needs to be determined, as

immortalized cell lines may exhibit properties different from

those of the living animal. In addition, cell lines lack the

innumerable regulatory systems that act directly or indirectly on

GnRH cells in vivo. To address the question of whether or not

GnRH cells in adult mice express Per1 message, we used micewith a green fluorescent protein (GFP) reporter driven by Per1

promoter. In these mice, GFP expression was observed in

neurons near to GnRH-immunoreactive cells, but the two

proteins were not co-localized. We have confirmed these

negative findings using double-label immunofluroscence in

mice and rats using several different antibodies against Per1 and

GnRH (Kriegsfeld and Silver, unpublished data). We conclude

that there are key differences between clock gene expression

between cultured GT1 cells and GnRH neurons in the brains of

adult animals.

System-level control and coordination of endocrine function

The task in understanding the orchestration of hormonal

systems is best understood by recognizing that most processes

in the body exhibit a circadian rhythm, and that activity in

various systems exhibits different phases (i.e., peak and trough

times) relative to each other. One explanation for how this feat is

accomplished suggests that the SCN secretes one neurochem-

ical to control each rhythmic process at its appropriate phase. In

this view, the SCN secretes numerous substances, each

precisely timed. Alternatively, control may be accomplished

by the specificity of SCN projections and combinations of

transmitter release at each target (Kalsbeek and Buijs, 2002). As

an alternative hypothesis, we have suggested that SCN timing

Table 1

List of mammalian neuroendocrine factors containing an E-box (CACGTG)

enhancer in their promoter

Gene E-box (#) Reference

Hypothalamic factors:CRH No (Muglia et al., 1994)

GHRH Yes (1) (Laird, 2001direct submission)

GnRH-I No (Hayflick et al., 1989)

GnRH-II No (White et al., 1998)

Ghrelin No (Kanamoto et al., 2004)

Oxytocin No (Hara et al., 1990)

Vasopressin Yes (1) (Hara et al., 1990; Jin et al., 1999)

TRH No (Satoh et al., 1996)

Pituitary:

POMC (ACTH, MSH) No (Drouin et al., 1985)

FSH No (Kumar, 1994direct submission)

GH No (Das et al., 1996)

LH No (Kaiser et al., 1998)

Prolactin No (Gubbins et al., 1980)

TSH (beta subunit) Yes (3) (Croyle and Maurer, 1984)



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signals have different consequences at each targeted effector

system (Kriegsfeld et al., 2003).

According to this view, a small number of rhythmic SCN

signals must be differentially interpreted by a large number of

targets to accomplish precise phase control by the SCN over an

ensemble of rhythmic processes. Different targets respond

differentially to the same message based on time of day (or localconditions), with some systems responding maximally to a

signal(s) at a particular time of day, while another system might

respond to this same signal(s) with inhibition. This hypothesis

requires that target systems of the SCN have a mechanism for

keeping time.

Seasonal as well as circadian timing are dependent upon the

SCN. Several hypotheses have been proposed to account for

how the SCN and its targets track time on a seasonal basis (Carr

et al., 2003; Hofman, 2004; Lincoln et al., 2003). In the SCN of

Syrian and Siberian hamsters, photoperiod alters the duration of

clock and clock-controlled gene expression, while the amplitude

of gene expression is influenced by photoperiod in the parstuberalis (Johnston et al., 2003; Messager et al., 2000). In sheep,

however, the relative timing of clock genes is altered by

photoperiod in the pars tuberalis, providing a mechanism of

temporal encoding and downstream control (Hazlerigg et al.,

2004; Lincoln et al., 2002, 2003, 2005). These correlational

results are intriguing and suggest that phase and/or amplitude of

clock and CCGs in SCN brain targets and endocrine glands may

predict their responsiveness to upstream signals on a daily

schedule.

Numerous systems display time-gated sensitivity to stimuli,

with the same stimulus or neurochemical producing different

effects at different times of day, suggesting temporal control at

effector sites rather than passive regulation by the SCN. Thepreoptic area, for example, exhibits robust clock gene

expression (Palm et al., 2001a,b; Tei et al., 1997; Yamamoto

et al., 2001. Importantly, stimulation of the POA of ovariecto-

mized rats with the SCN peptide, vasopressin, induces an LH

surge during the second portion of the light period, but not the

first (Palm, 2001 #122-001), indicating important temporal

control at the level of the POA.

Clocks in the neuroendocrine system

The fact that time-keeping machinery is functional in

numerous central neurosecretory cells (Kriegsfeld et al., 2003)and peripheral endocrine tissues (Bittman et al., 2003; Morse et

al., 2003; Shieh, 2003; Von Gall et al., 2002; Zylka et al., 1998)

represents a mechanism by which SCN targets can anticipate the

reception of SCN signals and respond based upon local needs

and time of day. In addition, because peripheral systems are

controlled hierarchically by multiple upstream components,

temporal modification in each link along the hypothalamo

pituitaryendocrine gland axis could provide additional control

over daily patterns of individual rhythms. At the top of this

circadian hierarchy of control, the SCN sends signals to

neuroendocrine cells and tissues to maintain synchronization

among cellular oscillators in phenotypically distinct neuroen-

docrine cell populations. Although capable of oscillating

independently, these neuroendocrine cells respond to periodic

SCN input to maintain a synchronized rhythm in the local cell

population. Such mechanism could account for the loss of

coherent circadian endocrine rhythms following lesions or

transection of outputs from the SCN (Meyer-Bernstein et al.,

1999; Moore and Eichler, 1972; Nunez and Stephan, 1977).

Loss of coherence among elements would result in a bluntedoverall output and absence of rhythmicity when individual cells

drift out of phase with each other.

If the SCN coordinates cell populations, why do individual

cells need their own clocks? According to this view, clocks in

SCN target populations provide responsiveness to local

conditions and temporal fine-tuning in a local population, not

possible with a single driving master clock. These local clocks

could selectively respond depending on time of day and

appropriately drive the expression of cell/tissue-dependent

CCGs that act as output to affect target systems or regulate

local conditions. Coordinated rhythmic output from neuroen-

docrine cells can then be communicated to the pituitary, whichalso exhibits circadian clock gene expression indicating a zone

for further temporal modification (Messager et al., 2000).

Finally, rhythmic information from the pituitary can be

communicated humorally to target glands in the periphery that

themselves express clock genes (Bittman et al., 2003; Zylka et

al., 1998). As mentioned previously, multisynaptic projections

from the SCN to several endocrine glands have been identified

using viral tracers (Buijs et al., 1998, 1999; Gerendai and

Halasz, 2000; Kalsbeek et al., 2000). These connections provide

a mechanism for the SCN to coordinate peripheral cellular

oscillators to optimize responses to slower endocrine signals.

Several lines of evidence support the notion that neural

communication from the SCN to the periphery is responsiblefor the timing of clock gene expression in targets organs and

glands (Guo et al., 2005; Shibata, 2004; Terazono et al., 2003).

The top of the hierarchy: neural SCN output

An organization of this type requires that the SCN

communicate (directly or indirectly) with neuroendocrine cells

expressing clock genes. There is substantial evidence of direct

projections from the SCN to neuroendocrine cells (Buijs et al.,

1993; Horvath et al., 1998; Kriegsfeld et al., 2002a,b;

Teclemariam-Mesbah et al., 1997; Van der Beek et al., 1997a,

b; Vrang et al., 1995). Expression of the clock gene, Per1, isrhythmically expressed in the Arc (Abe et al., 2002) and

exhibits a stress-induced increase in the PVH (Abe et al., 2002;

Takahashi et al., 2001). Per1 has been localized to CRH-ir cells

in the PVH (Takahashi et al., 2001), while the neurochemical

phenotype of Per1-expressing cells in the Arc has been

identified as dopaminergic providing a potential mechanism

of control of prolactin rhythms and the preovulatory prolactin

surge (Kriegsfeld et al., 2003).

Hormonal and neural communication to glands

Relative to the neural communication by the SCN to

neuroendocrine cells, hormonal communication is slow.



12/18

However, by using the bloodstream as a route of communica-

tion, hormones modulated by the circadian system can

communicate rhythmic information throughout the body.

Additional temporal control occurs at target glands and organs

by using circadian clock machinery to modulate responsiveness

to hormonal signals. If this means of temporal control is

implemented at peripheral targets, necessary alterations in thetiming of organ/gland responsiveness due to changes in local

conditions can be communicated rapidly to hormone-sensitive

targets to adjust the timing of their circadian clocks. Given that

numerous organs (e.g., liver, pancreas) and endocrine glands

(e.g., testes, adipose tissue, adrenal gland) investigated to date

receive autonomic innervation from the SCN (Bamshad et al.,

1998; Bartness et al., 2001; Buijs et al., 1999, 2003; Kalsbeek et

al., 2000; Olcese et al., 2003), these multisynaptic connections

may provide a rapid means of clock resetting in peripheral

tissues to ensure proper reception of slower diffusible

communication (Fig. 6). A role of autonomic control in

peripheral clock resetting comes from investigations in whichmanipulations of autonomic connections to liver reset clock

gene expression in this organ (Terazono et al., 2003).

In summary, global clock resetting may be accomplished via

hormonal signals, as glucocorticoids can adjust the phase of

peripheral circadian clock genes (Balsalobre et al., 2000).

Whereas the role of hormones other than glucocorticoids in

resetting peripheral oscillators has not been investigated, the

marked effects of hormones on circadian function reviewed

herein suggest a potentially crucial role for endocrine factors in

orchestrating this multioscillatory arrangement.

Conclusions and perspectives

In general, we are not aware of the precision in the timing

and coordination of numerous events in our bodies, unless it is

disrupted (e.g., jet lag). However, processes as fundamental as

the timing of sleep and its coordination with feelings of hunger

are a manifestation of numerous physiological and biochemicalevents that change systematically and predictably over the

course of the day. Given the numerous salient time cues in the

environment, one might intuit that these daily changes are

passive responses to environmental change. However, as

reviewed here, daily rhythms are endogenously generated and

are synchronized to external time cues in order to ensure that

bodily processes are carried out at the appropriate, optimal time

of day or night.

Because most brain and bodily processes require a

significant amount of time to achieve appropriate regulation

the body must anticipate these changes and prepare

accordingly in advance. For example, genomic actions ofsteroid hormones can take several hours to have their effects,

and these hormones must be secreted prior to the time during

which the behavior is best performed. Likewise, timed peak

and trough hormone secretion may be required to prevent

receptor down-regulation and desensitization. Because gener-

ating new receptors requires significant metabolic energy and

time, episodic hormone secretion may be required to allow for

adequate receptor turnover. Thus, an endogenous time-

keeping system is necessary to anticipate environmental

change and initiate internal adjustments in advance of the

Fig. 6. Overall organization of the circadian system. This organization is based on the postulation that rhythmic system physiology is controlled by a combination of

neural and diffusible signals originating from the SCN. In this view, specific systems may be differentially regulated by SCN signals via local clocks allowing for morespecific responsiveness based upon local needs and time of day (see text for additional details).



13/18

appropriate environmental time in order to coordinate

innumerable bodily processes.

The present overview shows how the circadian system

controls the timing of hormone secretion using a number of

mechanisms, including direct transcriptional and SCN neural

control of neurosecretory factors, and control of glands by

hormones, clock genes, and autonomic innervation. Becausehormones can have a widespread influence over physiology and

behavior, and provide a means by which circadian information

can be communicated systemically, it is important to determine

how these rhythms are regulated. Not only are hormones

modulated by the circadian system, but hormonal feedback to

the SCN also influences circadian function (Dubocovich et al.,

1996; Ellis and Turek, 1983; Hastings et al., 1997; Jechura et al.,

2003; Labyak and Lee, 1995; Lewy and Sack, 1997; Morin et al.,

1977). Together, these mechanisms controlling endocrine timing

entail regulatory actions by the circadian system and provide

extensive opportunities for empirical investigations of behav-

iorally relevant systems.

Acknowledgments

We thank Dr. Lily Yan for the discussions and suggestions

during the preparation of this review. We also thank Sean Duffy

for the editorial and technical assistance. The work described in

our laboratory was supported by NIH grants NS37919 (RS) and

MH-12408 (LJK).

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